Volume 16, Issue 12, Pages 3146-3156 (September 2016) Mammary-Stem-Cell-Based Somatic Mouse Models Reveal Breast Cancer Drivers Causing Cell Fate Dysregulation  Zheng Zhang, John R. Christin, Chunhui Wang, Kai Ge, Maja H. Oktay, Wenjun Guo  Cell Reports  Volume 16, Issue 12, Pages 3146-3156 (September 2016) DOI: 10.1016/j.celrep.2016.08.048 Copyright © 2016 The Author(s) Terms and Conditions

Cell Reports 2016 16, 3146-3156DOI: (10.1016/j.celrep.2016.08.048) Copyright © 2016 The Author(s) Terms and Conditions

Figure 1 Rapid Generation of Somatic GEMMs for Breast Cancer by Ex Vivo Expansion and Modification of MaSCs (A) Long-term expansion of MaSC organoids. Left: a representative image of organoids. Right: growth curves of two single cell-derived organoid clones. (B) CD49f, CD61, and PROCR flow cytometric profiles of organoids. (C) SLUG, SOX9, and cytokeratin immunostaining in organoids. The arrows indicate examples of SLUG+SOX9+ cells. (D) Whole-mount images of mammary ductal trees regenerated by single cell-derived GFP+ organoids at the indicated passages. (E) Immunofluorescence images of mammary ducts (left, virgin) and alveoli (right, pregnant) regenerated by organoids (passage 2). (F) Flow cytometric profiles of mammary ductal trees regenerated by organoids (passage 8). (G) A representative H&E image of poorly differentiated adenocarcinoma developed in Erbb2/MYC MaSC-GEMMs (with passage 3 organoids). Organoids were transplanted into non-obese diabetic (NOD)-severe combined immunodeficiency (SCID) mice, and the mice were treated with doxycycline for 4 months. (H) Representative images of carmine-stained mammary fat pads reconstituted by Rosa-CreERT2; Pik3ca∗ organoids (passages 3–5). Mice were treated with tamoxifen 6 weeks after transplantation and then analyzed 6 weeks later. The inset shows H&E staining of the outgrowths. (I) Representative images of carmine-stained mammary fat pads reconstituted by Blg-Cre; Brca1floxed/floxed; p53–/– organoids (passage 5). The inset shows H&E staining of the outgrowths. See also Figure S1. Cell Reports 2016 16, 3146-3156DOI: (10.1016/j.celrep.2016.08.048) Copyright © 2016 The Author(s) Terms and Conditions

Figure 2 Identification of Cancer Drivers by shRNA Screening in MaSC-GEMMs (A) Kaplan-Meier survival analysis of tumor onset in Pik3ca∗ MaSC-GEMMs expressing the indicated shRNAs (n = 14–40). ∗∗p < 0.01, ∗∗∗∗p < 0.0001, compared with shCtrl by the log-rank test. (B) Tumor growth rates of MaSC-GEMMs as shown in (A). Data are represented as mean ± SEM (n = 7–13). (C) Percentages of tumors with various histological phenotypes in the indicated MaSC-GEMMs (n = 11–20). ∗∗p < 0.01, chi-square test. (D) Lung metastases in the Pik3ca∗ MaSC-GEMMs expressing the indicated shRNAs. Left: representative H&E images of lung sections. Right: number of lung macrometastases per animal at the end point (mean ± SEM, n = 7–16). The frequency of mice with macrometastasis is shown on each column. (E) Growth curve of HCC1806 xenografts expressing doxycycline (dox)-inducible PTPN22 or the control vector. 100,000 cells were orthotopically injected into NOD-SCID mice. Three weeks after tumor onset, mice were treated with dox and measured for tumor volume. Each curve represents one tumor (n = 8, two-way ANOVA). (F) Lung metastasis burden of mice transplanted with Luc2-labeled HCC1806 cells as measured by bioluminescence. Cells and mice were treated as in (E) (n = 5, two-way ANOVA). (G) Relative tumor volume of animals treated by GDC-0941. Each dot represents the ratio of average volume of GDC-0941-treated (for ∼2 weeks) tumors to the average volume of vehicle-treated tumors that were derived from the same parental spontaneous tumor (n = 6–8). The tumors were either first- or second-generation allografts. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001. See also Figure S2. Cell Reports 2016 16, 3146-3156DOI: (10.1016/j.celrep.2016.08.048) Copyright © 2016 The Author(s) Terms and Conditions

Figure 3 Dysregulation of the Mammary Cell Fate by Ptpn22 Suppression (A) Whole-mount images of carmine-stained mammary fat pads transplanted with organoids expressing the indicated shRNAs (n = 6 for each group). Fat pads were isolated from mice in estrus ∼20 weeks post-transplantation. (B) H&E images of outgrowths as generated in (A). (C) Ki-67 immunohistochemistry (IHC) of outgrowths as generated in (A). Percentages of Ki-67+ cells in the mammary epithelium of individual outgrowths (minimum 6 fields/outgrowth) are shown (n = 4–5). (D) Cytokeratin immunostaining of mammary outgrowths as generated in (A). (E) Flow cytometric analysis of mammary outgrowths formed by organoids expressing the control or Ptpn22 shRNAs. Glands were analyzed 12 weeks post-transplantation. The graph shows percentages of CD61high cells in lineage-negative epithelial cells (mean ± SEM, n = 4–11). (F) Organoid structures generated by the indicated cell populations (left) and quantification of solid organoid-forming ability (mean ± SEM) (right). (G) Organoid-forming efficiency (mean ± SEM) of the indicated cell types seeded in full organoid medium or medium without FGF2. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S3. Cell Reports 2016 16, 3146-3156DOI: (10.1016/j.celrep.2016.08.048) Copyright © 2016 The Author(s) Terms and Conditions

Figure 4 CRISPR-Mediated MaSC-GEMMs Reveal the Role of Mll3 in Mammary Tumorigenesis and Stem Cell Regulation (A) RFLP assay for determining the Mll3 genotype of single cell-derived organoid clones. The wild-type amplicon can be digested by TaiI. (B) MLL3 protein levels in organoid clones as determined by western blot. (C) Whole-mount images of cleared mammary fat pads transplanted by Mll3+/+ (vector control) or Mll3–/– (clone #2) organoids. Glands were analyzed 12 weeks post-transplantation. (D) H&E images of outgrowths as generated in (C) (E) KRT14 and KRT8 immunofluorescence of outgrowths as generated in (C). (F) Flow cytometric analysis of outgrowths generated by organoids expressing the control or Mll3 lentiCRISPRv2 vector (top). The percentages of stem/basal (B/S), luminal progenitor (LP), and mature luminal cells (ML) in lineage-negative epithelial cells are shown (mean ± SEM, n = 3–4) (bottom). (G) Organoid-forming efficiency of Mll3+/+ (vector control) and Mll3–/– organoid cells. Each data point represents an independent organoid line (mean ± SEM, n = 3–4). (H) Organoid-forming efficiency (mean ± SEM) of cells transduced by the indicated shRNAs. (I) Kaplan-Meier survival analysis of PIK3CA∗-driven tumor onset in Mll3+/+ or Mll3–/– MaSC-GEMMs. p values were determined by log-rank test. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. ns, not significant. See also Figure S4. Cell Reports 2016 16, 3146-3156DOI: (10.1016/j.celrep.2016.08.048) Copyright © 2016 The Author(s) Terms and Conditions

Figure 5 Activation of the HIF Pathway Mediates the Effect of Mll3 Deletion on Stem Cell Activation (A) Two representative gene sets upregulated in Mll3–/– organoids as determined by GSEA. (B) Relative mRNA levels of HIF target genes in Mll3+/+ and Mll3–/– organoid lines (n = 3) as measured by qRT-PCR. Gapdh and Hprt were used as internal controls. (C) HIF-1α protein levels in two independent lines of Mll3+/+ (vector control) and Mll3–/– organoids as measured by western blot. Cells were transduced by the lentiCRISPRv2 vectors. Histone H3 was used as a loading control. (D) Effect of the HIF inhibitor acriflavine (ACF) on the organoid-forming ability of Mll3+/+ and Mll3–/– cells. Cells were cultured with the indicated concentration of ACF for 7 days. Organoid-forming efficiency was normalized to the respective DMSO control. (E) Effect of hypoxia on organoid-forming ability. Mll3+/+ and Mll3–/– cells were cultured at the indicated oxygen concentrations for 7 days. The data are represented as mean ± SEM. See also Figure S5. Cell Reports 2016 16, 3146-3156DOI: (10.1016/j.celrep.2016.08.048) Copyright © 2016 The Author(s) Terms and Conditions